U.S. patent application number 16/618118 was filed with the patent office on 2020-06-11 for cd8 t cell subsets as a biomarker for predicting non-fusion after spinal fusion surgery.
This patent application is currently assigned to CHARITE UNIVERSITATSMEDIZIN BERLIN. The applicant listed for this patent is CHARITE UNIVERSITATSMEDIZIN BERLIN. Invention is credited to Georg DUDA, Michael FUCHS, Sven GEISSLER, Matthias PUMBERGER, Simon REINKE, Katharina SCHMIDT-BLEEK, Patrick STRUBE, Hans-Dieter VOLK.
Application Number | 20200182872 16/618118 |
Document ID | / |
Family ID | 62200471 |
Filed Date | 2020-06-11 |
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United States Patent
Application |
20200182872 |
Kind Code |
A1 |
REINKE; Simon ; et
al. |
June 11, 2020 |
CD8 T CELL SUBSETS AS A BIOMARKER FOR PREDICTING NON-FUSION AFTER
SPINAL FUSION SURGERY
Abstract
The present invention relates to a method for predicting the
probability of having or developing a non-fusion, wherein said
method comprises determining the frequency of a subpopulation of
CD8+ T cells selected from CD8+CD57+, CD8+CD28- and CD8+CD57+CD28-
in a sample obtained from a patient. The invention further relates
to a system for predicting the probability of having or developing
a non-fusion.
Inventors: |
REINKE; Simon; (Berlin,
DE) ; GEISSLER; Sven; (Berlin, DE) ; DUDA;
Georg; (Berlin, DE) ; VOLK; Hans-Dieter;
(Berlin, DE) ; FUCHS; Michael; (Berlin, DE)
; SCHMIDT-BLEEK; Katharina; (Berlin, DE) ; STRUBE;
Patrick; (Jena, DE) ; PUMBERGER; Matthias;
(Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHARITE UNIVERSITATSMEDIZIN BERLIN |
Berlin |
|
DE |
|
|
Assignee: |
CHARITE UNIVERSITATSMEDIZIN
BERLIN
Berlin
DE
|
Family ID: |
62200471 |
Appl. No.: |
16/618118 |
Filed: |
May 29, 2018 |
PCT Filed: |
May 29, 2018 |
PCT NO: |
PCT/EP2018/064085 |
371 Date: |
November 28, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/70521
20130101; C07K 14/51 20130101; G01N 2333/70517 20130101; G01N
2800/10 20130101; G01N 2333/70596 20130101; A61K 38/00 20130101;
C12N 2501/155 20130101; G01N 2800/52 20130101; G01N 33/56972
20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C07K 14/51 20060101 C07K014/51 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 2017 |
EP |
17173320.7 |
Dec 12, 2017 |
EP |
17206698.7 |
Claims
1. Method for predicting the probability of having or developing a
non-fusion, particularly before undergoing a spinal fusion surgery
or after a spinal fusion surgery, wherein said method comprises
determining the frequency of a subpopulation of CD8+ T cells
selected from CD8+CD57+, CD8+CD28- and CD8+CD57+CD28- in a sample
obtained from a patient.
2. Method for monitoring the course of treatment of a non-fusion in
a patient, particularly after spinal fusion surgery, wherein said
method comprises determining the frequency of a subpopulation of
CD8+ T cells selected from CD8+CD57+, CD8+CD28- and CD8+CD57+CD28-
in a sample obtained from said patient.
3. The method according to claim 1, wherein said non-fusion is a
non-fusion between two or more bones or bone segments naturally not
connected by osseous tissue, particularly a non-fusion between two
or more vertebrae.
4. The method according to claim 1, wherein said sample is a blood
sample.
5. The method according to claim 1, wherein said sample is a sample
that has been obtained before, during or after spinal fusion
surgery from said patient.
6. The method according to claim 1, wherein a sample exhibiting an
at least two-fold higher frequency of said subpopulation of CD8+ T
cell when compared to a standard value determined for a large
population with normal fusion after bone fusion is assigned to a
patient having an elevated probability for having or developing
non-fusion after spinal fusion surgery.
7. The method according to claim 1, wherein a sample exhibiting a
fraction of CD8+CD57+CD28- cells referred to the total sum of all
CD8+ cell in said sample of at least 22.7% more particular of at
least 33.1%, is assigned to a patient having an elevated
probability for having or developing non-fusion after spinal fusion
surgery, and/or a sample exhibiting a summed fraction of CD8+CD57+
cells and CD8+CD28- cells referred to the total sum of all CD8+
cells in said sample of at least 85.6% is assigned to a patient
having an elevated probability for having or developing non-fusion
after spinal fusion surgery, and/or a sample exhibiting a fraction
of CD8+CD57+ cells referred to the total sum of all CD8+ cells in
said sample of at least 24.4%, more particular of at least 37.6%,
is assigned to a patient having an elevated probability for having
or developing non-fusion after spinal fusion surgery, and/or a
sample exhibiting a fraction of CD8+CD28- cells referred to the
total sum of all CD8+ cells in said sample of at least 28.0%, more
particular of at least 42.9%, is assigned to a patient having an
elevated probability for having or developing non-fusion after
spinal fusion.
8. A system for predicting the probability of having or developing
a non-fusion, particularly before undergoing a spinal fusion
surgery or after a spinal fusion surgery, and/or for monitoring the
course of treatment of a non-fusion of a patient, particularly
after spinal fusion surgery, comprising a device for determination
the frequency of a cell population in a sample obtained from a
patient programmed microprocessor, wherein said microprocessor is
configured to run a method according to claim 1.
9. Use of a combination of antibodies for predicting the
probability of having or developing a non-fusion, particularly
before undergoing a spinal fusion surgery after a spinal fusion
surgery, wherein said combination comprises an anti-CD8-antibody
and an anti-CD57-antibody, or an anti-CD8-antibody and an anti-CD28
antibody, or an anti-CD8-antibody, an anti-CD28-antibody and an
anti-CD57-antibody, and wherein said antibodies are suitable for
fluorescence based flow cytometry.
10. A bone morphogenetic protein for use in a method for preventing
or treating non-fusion after spinal bone fusion surgery, wherein
said bone morphogenetic protein is administered during or after
undergoing a spinal fusion surgery or a revision surgery after
failed spinal fusion to a patient that has an elevated probability
of developing or having a non-fusion after spinal bone fusion
surgery.
11. The bone morphogenetic protein for use in a method for treating
non-fusion after spinal bone fusion surgery in a patient having an
elevated probability of non-fusion after spinal bone fusion surgery
according to claim 10, wherein said bone morphogenetic protein is
selected from BMP2, or BMP7,
12. The bone morphogenetic protein for use in a method for treating
non-fusion after spinal bone fusion surgery in a patient having an
elevated probability of non-fusion after spinal bone fusion surgery
according to claim 10, wherein said elevated probability of
developing or having a non-fusion after spinal bone fusion surgery
have been determined by a method according to claim 1.
Description
[0001] The present invention relates to a method for predicting or
detecting non-fusion, particularly before undergoing or after a
spinal fusion surgery.
BACKGROUND
[0002] Chronic low back pain are one of the major reasons for
disability or early work retirement in the industrial world. There
are multiple reasons for the lower back pain (e.g. biological,
anatomical, degeneration) resulting in serious problems like
spondylisthesis, spondylosis, osteochondrosis or spinal stenosis.
After an intensive long term conservative treatment with
physiotherapy most of these affected patients have to undergo a
spinal bone fusion surgery in terms of arthrodesis to relieve
intractable pain.
[0003] Between 1998 and 2008, the annual number of spinal fusion
surgeries in the US increased 2.4-fold (137%) from 174,223 to
413,171. [Rajaee S S et al.: Spinal fusion in the United States:
analysis of trends from 1998 to 2008. 2012 Jan. 1; 37(1):67-76]. In
2011, 488,000 spinal fusion surgeries were reported in the United
States of America, representing 3.1% (6.sup.th rank) all
residential treatments. Moreover, with around 27,000$ per case, the
surgical treatment of spinal fusion patient is most
expensive[Agency for Healthcare Research and Quality (AHRQ)]. In
Germany, a total of approx 230.000 spinal operations were conducted
in 2011 representing an increase of 136% compared to 2008
[Deutsches Arzteblatt]
[0004] Fusion rates following the surgical treatment for spinal
fusion surgery shows a wide range of successful de novo bone
formation from 46% to almost 100%. Based on the FDA guidelines, a
delayed union is characterized "when healing has not advanced at
the average rate for the location between 3-6 months". A non-fusion
(pseudarthrosis) is defined by a "healing time of >9 month with
no radiographic progression for 3 month." There are several factors
that affect the success of fusion like patient co-morbidities,
implant position and biologic deficiencies. Moreover, the various
available bone graft materials can also affect the outcome of
spinal fusion. Normally, the most used graft material comes from
iliac crest bone.
[0005] However, assessing the patients function and quality of
life, high rates of post-operative pain and morbidity often result
from this additional procedure. Together with the abovementioned
limited fusion rates due the biologic deficiencies of the patient
this represents a serious medical problem with a high
socio-economic impact. The usage of bone morphogenetic protein
(BMP-2), is so far authorized in a revision scenario only, due to
high costs and different side effects. Chun et al. reported in
2015: "The best treatment for pseudarthrosis is to prevent it from
occurring after the initial operation". [Chun D S et al.; Lumbar
pseudarthrosis: a review of current diagnosis and treatment.
Neurosurg Focus. 2015 October; 39(4).]
[0006] Therefore, there is an unmet need for predicting the outcome
after spinal fusion surgery to personalize as early as possible the
therapeutic use of supporting growth factors, like BMP's, which are
costly and not free of adverse effects.
[0007] It is therefore the objective of the present invention to
provide means and methods for an accurate prediction or detection
of non-fusion, particularly before or after a spinal fusion
surgery.
DESCRIPTION OF THE INVENTION
[0008] The above objective is attained by a method having the
features of claim 1 or 2 and by a system having the features of
claim 7. Preferred embodiments are state in the sub claims and the
description below.
[0009] According thereto, a first aspect of the invention relates
to a method for predicting the probability of having or developing
a non-fusion, particularly before undergoing a spinal fusion
surgery or before revision surgery in failed spinal fusion or after
a spinal fusion surgery. The method comprises determining the
frequency of a subpopulation of CD8+ cells or CD8+CD3+CD45+ cells
selected from CD8+CD57+, CD8+CD28- and CD8+CD57+CD28- in a sample
obtained from a patient.
[0010] During the course of investigation of non-fusion after
spinal fusion, it was surprisingly found that the spinal non-fusion
was accompanied by a significantly elevated frequency of terminally
differentiated CD8+ effector T cells expressing the phenotype
CD45+CD3+CD8+CD28- or CD45+CD3+CD8+CD57+ or CD45+CD3+CD8+CD57+CD28-
(CD8+ TEMRA). These cells are characterized by their tissue homing
properties and strong bystander activity. They can be triggered
independent on their T-cell receptor in an antigen-independent
matter by cytokines delivered from cells of innate immunity (like
macrophages or dendritic cells) triggered within the inflamed
tissue as result of Toll Receptor (TLR). Damage-associated
molecular pattern (DAMPs) interaction (e.g. IL-12, IL-18) can
trigger the release of inflammatory cytokines (e.g. IFN-gamma) by
these CD8+ TEMRA that support overwhelming inflammation and
fibrosis. In contrast to other T cells, these CD8+ TEMRA do not
need T-cell receptor (TCR)-crosslinking for activation.
[0011] If any cell population is designated "positive" with respect
to a certain marker molecule herein, this designation shall
particularly mean that said cell population can be stained by a
common fluorescent-dye-labelled antibody against the marker
molecule and will give a fluorescence signal of at least one, two
or three log higher intensity compared to unlabeled cells or cells
labelled with the same antibody but commonly known as not
expressing said marker molecule or an isotype control antibody.
Vice versa, any cell population that is designated "negative" with
respect to certain marker molecule cannot be stained by a
fluorescent-dye labelled antibody as described above against the
marker molecule.
[0012] A second aspect of the invention relates to a method for
monitoring the course of treatment of a non-fusion, particularly
after spinal fusion surgery. The method comprises determining the
frequency of a subpopulation of CD8+ T cells or CD8+CD3+CD45+ cells
selected from CD8+CD57+, CD8+CD28- and CD8+CD57+CD28- in a sample
obtained from said patient.
[0013] The term "non-fusion" or "nonunion" is used in its meaning
known in the field of medicine, more particular in the field of
orthopaedic surgery. It particularly refers to the absence of a
consolidation of two bone segments within 9 months with no
radiographic progression for 3 months, wherein particularly the two
bone segments are naturally not connected by osseous tissue.
Nonunion or non-fusion is also referred to as pseudoarthrosis.
[0014] Advantageously in case of revision surgery in failed spinal
fusion, low doses of, e.g., BMP can be administered to a patient
with a confirmed high risk of having or developing non-fusion.
[0015] In certain embodiments of the first or second aspect of the
invention, the non-fusion is a non-fusion between two or more bones
or bone segments naturally not connected by osseous tissue.
[0016] In certain embodiments of the first or second aspect of the
invention, the non-fusion is a non-fusion between two or more
vertebrae.
[0017] In certain embodiments of the first or second aspect of the
invention, the sample is a blood sample, particularly a sample from
the peripheral blood.
[0018] In certain embodiments of the first or second aspect of the
invention, the sample is a sample that has been obtained from the
patient before, during or after the spinal fusion surgery.
[0019] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting an at least 1.5 higher frequency of
the subpopulation of CD8+ T cells or CD8+CD3+CD45+ cells when
compared to a standard value determined for a large population with
normal fusion after spinal fusion surgery is assigned to a patient
having an elevated probability for having or developing non-fusion
after spinal fusion surgery.
[0020] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting an at least 1.8 higher frequency of
the subpopulation of CD8+ T cells or CD8+CD3+CD45+ cells when
compared to a standard value determined for a large population with
normal union after spinal fusion is assigned to a patient having an
elevated probability for having or developing non-fusion after
spinal fusion surgery.
[0021] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting an at least two-fold higher
frequency of the subpopulation of CD8+ T cells or CD8+CD3+CD45+
cells when compared to a standard value determined for a large
population with normal union after spinal fusion is assigned to a
patient having an elevated probability for having or developing
non-fusion after spinal fusion surgery.
[0022] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting a fraction of CD8+CD57+CD28- cells
referred to the total sum of all CD8+ cell in the sample of at
least 22.7% more particular of at least 33.1%, is assigned to a
patient having an elevated probability for having or developing
non-fusion after spinal fusion surgery.
[0023] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting a summed fraction of CD8+CD57 cells
and CD8+CD28- cells referred to the total sum of all CD8+ cells in
the sample of at least 85.6% is assigned to a patient having an
elevated probability for having or developing non-fusion after
spinal fusion surgery.
[0024] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting a fraction of CD8+CD28- cells
referred to the total sum of all CD8+ cells in the sample of at
least 28.0%, more particular of at least 42.9%, is assigned to a
patient having an elevated probability for having or developing
non-fusion after spinal fusion surgery.
[0025] In certain embodiments of the first or second aspect of the
invention, a sample exhibiting a fraction of CD8+CD57+ cells
referred to the total sum of all CD8+ cells in the sample of at
least 28.0%, more particular of at least 42.9%, is assigned to a
patient having an elevated probability for having or developing
non-fusion after spinal fusion.
[0026] In certain embodiments, the method according to the first or
second aspect of the invention is performed by contacting a sample
with a first ligand specifically reactive to a marker molecule
selected from a marker group comprised of CD8 (UniProt P01732,
P10966), CD28 (UniProt P10747) and CD57 (UniProt Q9P2W7), and
determining the frequency of a cell presenting the marker molecule
marked with the first ligand or determining the frequency of the
first ligand bound to the marker molecule.
[0027] In certain embodiments of the first or second aspect of the
invention, the first ligand is selected from the group comprised of
an antibody, an antibody fragment, an antibody-like-molecule, an
oligopeptide of 6 to 30 amino acids, and a nucleic acid aptamer
molecule of 10 to 75 nucleotides in length, wherein the ligand is
capable to bind to a member of the marker group described in the
preceding paragraph with a dissociation constant of 10.sup.-8 mol/l
or smaller.
[0028] In certain embodiments of the first or second aspect of the
invention, the antibody fragment is the Fab domain of an antibody
(the antigen binding region of an antibody) or a single chain
antibody (scFv), a fusion protein consisting of the variable
regions of light and heavy chains of an antibody connected by a
peptide linker. An antibody fragment or an antibody-like molecule
may be manufactured by suitable methods such as recombinant protein
expression.
[0029] Such first ligand may also be developed by evolutive methods
such as phage display, ribosome display or SELEX, wherein
polypeptides or oligonucleotides are selected according to their
binding affinity to a target of interest. Additionally, higher
affinity inhibitors may be identified by reiterative rounds of
evolution and selection of the amino acid sequence or nucleotide
sequence.
[0030] In certain embodiments of the first or second aspect of the
invention, the oligopeptide of 6 to 30 amino acids as referred to
above is a peptide derived from the part of a ligand, which is
recognized by a member of the marker group described above.
[0031] In certain embodiments of the first or second aspect of the
invention, the ligand that is recognized by a member of the marker
group described above is selected from CD80 (Uniprot ID P33681) or
CD86 (Uniprot ID P42081), which are ligands of CD28.
[0032] In certain embodiments of the first or second aspect of the
invention, the first ligand is an antibody reactive to CD8, CD28,
or CD57 and further comprises a fluorescent moiety for optical
detection, wherein a marker molecule according to the preceding
paragraphs is bound to such antibody, and cells presenting that
marker molecule can be counted by a fluorescence based flow
cytometric method such as fluorescence activated cell sorting.
[0033] In certain embodiments of the first or second aspect of the
invention, the first ligand is specifically bound by a second
ligand, wherein the second ligand comprises an enzymatic activity
or a fluorescent moiety. A plurality of different marker molecules
may be determined by use of a plurality of different first ligands,
wherein each ligand specifically binds to a particular marker
molecule.
[0034] In certain embodiments of the first or second aspect of the
invention, each first ligand of the plurality comprises a certain
enzymatic activity or fluorescent moiety as described above that
can be spectroscopically distinguished form the enzymatic activity
or fluorescent moiety of each other first ligand of the
plurality.
[0035] In certain embodiments of the first or second aspect of the
invention, each first ligand is bound be a specific second ligand
having a certain enzymatic activity or fluorescent moiety that can
be spectroscopically distinguished for the enzymatic activity or
fluorescent moiety of each other second ligand.
[0036] In certain embodiments of the first or second aspect of the
invention, the first and second ligand or pluralities thereof are
antibodies and used in an Enzyme-linked immunosorbent assay.
[0037] In certain embodiments of the first or second aspect of the
invention, the frequency of a subpopulation according to the
preceding aspect of the invention, particularly of CD8+CD57+,
CD8+CD28- or CD8+CD57+CD28- T cells, is determined by counting
cells that are marked with a fluorescent antibody directed to a
marker molecule selected from group comprised of CD8, CD28 and CD57
in a flow cytometric assay.
[0038] In certain embodiments of the first or second aspect of the
invention, the antibodies according to the above aspects or
embodiments of the invention are monoclonal antibodies of murine
origin and comprise a fluorescent moiety for optical detection in
the flow cytometry such as APC (allophycocyanin), FITC (fluorescein
isothiocyanate) or PE (phycoerythrin).
[0039] In certain embodiments of the first or second aspect of the
invention, the antibody is selected from the group comprised of
APC-Cy7-labelled mouse anti-human CD8 IgG1, a murine monoclonal
antibody conjugated with the tandem fluorophor APC-Cy7,
FITC-labelled mouse anti-human CD57 IgM, a murine monoclonal
antibody, and APC-H7 mouse anti-human CD28 IgG1, a murine
monoclonal antibody conjugated with the tandem fluorophor APC-H7
being an analogue of APC-Cy7 and having the same spectroscopic
properties.
[0040] According to another aspect of the invention, the use of a
combination of antibodies for predicting the probability of having
or developing a non-fusion, particularly after spinal fusion
surgery is provided, wherein the combination comprises an
anti-CD8-antibody and an anti-CD57-antibody, or an
anti-CD8-antibody and an anti-CD28 antibody, or an
anti-CD8-antibody, an anti-CD28-antibody and an anti-CD57-antibody,
and wherein said antibodies are suitable for fluorescence based
flow cytometry.
[0041] In certain embodiments of above aspect of the invention, the
antibodies according to the above aspect of the invention are
monoclonal antibodies of murine origin and comprise a fluorescent
moiety for optical detection in the flow cytometry such as APC
(allophycocyanin), FITC (fluorescein isothiocyanate) or PE
(phycoerythrin).
[0042] In certain embodiments of the above aspect of the invention,
the antibody is selected from the group comprised of
APC-Cy7-labelled mouse anti-human CD8 IgG1, a murine monoclonal
antibody conjugated with the tandem fluorophor APC-Cy7,
FITC-labelled mouse anti-human CD57 IgM, a murine monoclonal
antibody, and APC-H7 mouse anti-human CD28 IgG1, a murine
monoclonal antibody conjugated with the tandem fluorophor APC-H7
being an analogue of APC-Cy7 and having the same spectroscopic
properties.
[0043] According to a further aspect of the invention, a system for
predicting the probability of having or developing non-fusion,
particularly before undergoing a spinal fusion surgery, or for
monitoring the course of treatment of non-fusion, particularly
after spinal fusion surgery, of a patient is provided, comprising
[0044] a device for determination the frequency of a cell
population in a sample from a patient, and [0045] a programmed
microprocessor, wherein the programmed microprocessor is configured
to run a method according to the above aspects or embodiments of
the invention.
[0046] In certain embodiments, the device is a flow cytometer,
comprising a flow cell for transporting and aligning cells, a light
source such as a laser and a detector suitable for measuring light
or other biophysical parameters such as impedance. Such device may
be used for determining the frequency of subpopulations of CD8+
cells according to the above aspects and embodiments of the
invention.
[0047] In certain embodiments, the device is spectrophotometer or a
plate reader, comprising a compartment holding the sample such as a
cuvette or a microtiter plate, a light source and a UV/Vis detector
suitable for measurement of absorbance or fluorescence, such as a
diode array.
[0048] In certain embodiments, the programmed microprocessor is
integrated in the device described in the preceding paragraphs or
is part of a control unit or a computer for operating the
device.
[0049] In certain embodiments, the device described above is
configured to determine the frequency of the subpopulation of CD8+
T cells or CD8+CD3+CD45+ cells according to the above aspects and
embodiments of the invention, particularly of CD8+CD57+, CD8+CD28-
or CD8+CD57+CD28- T cells.
[0050] According to yet another aspect of the invention, a bone
morphogenetic protein for use in a method for preventing or
treating non-fusion after spinal fusion surgery is provided,
wherein the bone morphogenetic protein is administered during or
after undergoing a spinal fusion surgery to a patient that has an
elevated probability of developing or having a non-fusion after
spinal bone fusion surgery.
[0051] In certain embodiments, the bone morphogenetic protein is
BMP2 or BMP7.
[0052] In certain embodiments, the elevated probability of
developing or having a non-fusion after spinal bone fusion surgery
have been determined by a method according to the first aspect or
any embodiment thereof.
SHORT DESCRIPTION OF THE FIGURES
[0053] FIG. 1 shows the results of flow cytometric analyses of the
circulating immune cell subset CD45+CD3+CD8+CD57+ T cells in the
peripheral blood of patients with spinal non-fusion and normal
fusion. Spinal non-fusion patients (healing class 2) showed a
significantly higher frequency of CD45+CD3+CD8+CD57+ T cells (CD57+
in % of CD45+CD3+CD8+ T cells) compared to normal healing patients
(healing class 1) indicates a dramatic increase of terminally
differentiated CD8+ effector cells (TEMRA CD8+). *=P<0.001;
Mann-Whitney U test.
[0054] FIG. 2 Results of flow cytometric analyses of the
circulating immune cell subset CD45+CD3+CD8+CD28- T cells in the
peripheral blood of patients with spinal non-fusion and normal
fusion. Spinal non-fusion patients (healing class 2) showed a
significantly higher frequency of CD45+CD3+CD8+CD28- T cells (CD28-
in % of CD45+CD3+CD8+ T cells) compared to normal healing patients
(healing class 1) indicates a dramatic increase of terminally
differentiated CD8+ effector cells (TEMRA CD8+). *=P<0.001;
Mann-Whitney U test.
[0055] FIG. 3 shows the results of flow cytometric analyses of the
circulating immune cell subset CD45+CD3+CD8+CD57+28- T cells in the
peripheral blood of patients with spinal non-fusion and normal
fusion. Spinal non-fusion patients (healing class 2) showed a
significantly higher frequency of CD45+CD3+CD8+CD57+28- T cells
(CD57CD28-+ in % of CD45+CD3+CD8+ T cells) compared to normal
healing patients (healing class 1) indicates a dramatic increase of
terminally differentiated CD8+ effector cells (TEMRA CD8+).
*=P<0.001; Mann-Whitney U test.
[0056] FIG. 4 illustrates the receiver operating characteristics
(ROC) based on the summation of the expression of CD57+ in % of
CD45+CD3+CD8+ T cells and CD28- in % of CD45+CD3+CD8+ T cells. The
ROC analysis showed a high sensitivity (76%) as well as specificity
(96%) in the detection of the affected patients based on their
blood levels using a cut-off level of 85.6% with an area under the
curve (AUC): 0.886 and p<0.001.
[0057] FIG. 5 illustrates the receiver operating characteristic
(ROC) based on the expression of CD57+CD28- in % of CD45+CD3+CD8+ T
cells with an area under the curve (AUC): 0.859 and p<0.001. The
ROC analysis showed a high sensitivity (90%) as well as a
specificity (71%) in the detection of the affected patients based
on their blood levels using a cut-off level of 22.7%. A cut-off
level of 33.1% showed a sensitivity of 71% with a higher
specificity (96%)
[0058] FIG. 6 illustrates the receiver operating characteristics
(ROC) based on the expression of CD57+ in % of CD45+CD3+CD8+ T
cells with an area under the curve (AUC): 0.863 and p<0.001. The
ROC analysis showed a high sensitivity (90%) as well as a
specificity (61%) in the detection of the affected patients based
on their blood levels using a cut-off level of 24.4%. A cut-off
level of 37.6% showed a sensitivity of 71% with a higher
specificity (96%)
[0059] FIG. 7 illustrates the receiver operating characteristics
(ROC) based on the expression of CD28- in % of CD45+CD3+CD8+ T
cells with an area under the curve (AUC): 0.876 and p<0.001. The
ROC analysis showed a high sensitivity (90%) as well as a
specificity (66%) in the detection of the affected patients based
on their blood levels using a cut-off level of 28.0%. A cut-off
level of 42.9% showed a sensitivity of 76% with a higher
specificity (91%)
EXAMPLES
[0060] In order to define novel biomarkers for predicting the
individual risk and to identify novel therapeutic targets, the
inventors included 44 patients that received a spinal bone fusion
surgery with secured vertebral body fusion results into a
retrospective study. These patients were classified in patients
with normal vertebral body fusion (n=23) and patients with
pseudarthrosis/non-fusion (n=21), based on their CT-scan
results.
[0061] Table 1 shows the characterization and blood parameters of
the patients involved in the retrospective study (spinal fusion and
spinal non-fusion patients)
TABLE-US-00001 Spinal non- Spinal fusion p- Parameter fusion (n =
21) (n = 23) value Age (y) 70.7 .+-. 8.1 63.7 .+-. 10.1 0.01 Sex 6
male/ 10 male/ 0.3 15 female 13 female (28.6%)/ (43.5%)/ (71.4%)
(56.5%) Time to revision or 21.7 .+-. 12.7 27.0 .+-. 10.3 0.1
revision free Minimum: 8 Minimum: 12 (month) Maximum: 47 Maximum:
57 Height (m) 1.62 .+-. 0.1 1.66 .+-. 0.1 0.3 Weight (kg) 76.9 .+-.
10.7 79.6 .+-. 13.7 0.5 BMI 29.1 .+-. 3.2 29.3 .+-. 6.4 0.9
Leucocytes (/nL) 8.5 .+-. 3.9 6.7 .+-. 2.0 0.06 Haemoglobin (g/L)
13.2 .+-. 1.9 14.2 .+-. 1.3 0.06 Haematocrite (%) 0.39 .+-. 0.05
0.41 .+-. 0.03 0.07 Erythrocytes (/pL) 4.4 .+-. 0.6 4.6 .+-. 0.5
0.2 Thrombocytes (/nL) 271.5 .+-. 77.8 253.0 .+-. 53.0 0.3 Sodium
(mmol/L) 139.0 .+-. 3.9 139.0 .+-. 2.9 0.9 Potassium (mmol/L) 4.2
.+-. 0.4 4.1 .+-. 0.3 0.6 Calcium (mmol/L) 2.4 .+-. 0.1 2.3 .+-.
0.1 0.4 Glucose (mg/dL) 102.3 .+-. 24.1 109.1 .+-. 41.8 0.5
Creatinin (mg/dL) 0.88 .+-. 0.2 0.81 .+-. 0.2 0.3 Urea (mg/dL) 32.8
.+-. 9.2 32.4 .+-. 11.3 0.9 Albumin (g/L) 42.9 .+-. 9.3 44.5 .+-.
2.7 0.4 GPT (U/L) 24.5 .+-. 19.9 28.4 .+-. 16.0 0.5 GOT (U/L) 26.5
.+-. 14.2 31.0 .+-. 28.8 0.5 CRP (mg/L) 0.9 .+-. 1.1 4.0 .+-. 6.0
0.03
[0062] Table 2: shows the blood parameters and time to diagnosis of
spinal non-fusion at the initial and revision surgery for
non-fusion patients.
TABLE-US-00002 Spinal non- fusion Initial Spinal non-fusion p-
Parameter Surgery Revision Surgery value Time to diagnose 8 month =
n = 2 = 9.5% non-fusion (month) 10 month = n = 2 = 9.5% 11 month =
n = 2 = 9.5% 12 month = n = 3 = 14.3% 13 month = n = 1 = 4.8% 18
month = n = 1 = 4.8% 19 month = n = 1 = 4.8% 24 month = n = 1 =
4.8% 26 month = n = 1 = 4.8% 31 month = n = 1 = 4.8% 33 month = n =
1 = 4.8% 36 month = n = 2 = 9.5% 40 month = n = 2 = 9.5% 47 month =
n = 1 = 4.8% Revision performed -- 15/21 (71.4%) -- Spongiosa +
BMP-2 -- 8/15(53.3%) -- Spongiosa 7/15 (46.7%) -- Leucocytes (/nL)
8.5 .+-. 3.9 6.5 .+-. 1.6 0.9 Haemoglobin (g/L) 13.2 .+-. 1.9 13.9
.+-. 1.9 0.6 Haematocrite (%) 0.39 .+-. 0.05 0.41 .+-. 0.06 0.8
Erythrocytes (/pL) 4.4 .+-. 0.6 4.4 .+-. 0.7 0.7 Thrombocytes (/nL)
271.5 .+-. 77.8 261.4 .+-. 68.1 0.5 Sodium (mmol/L) 139.0 .+-. 3.9
137.7 .+-. 4.4 0.4 Potassium (mmol/L) 4.2 .+-. 0.4 4.2 .+-. 0.4 0.7
Calcium (mmol/L) 2.4 .+-. 0.1 2.4 .+-. 0.1 0.5 Glucose (mg/dL)
102.3 .+-. 24.1 106.4 .+-. 23.8 0.3 Creatinin (mg/dL) 0.88 .+-. 0.2
0.87 .+-. 0.2 0.7 Urea (mg/dL) 32.8 .+-. 9.2 36.5 .+-. 9.1 0.3
Albumin (g/L) 42.9 .+-. 9.3 43.8 .+-. 2.9 0.4 GPT (U/L) 24.5 .+-.
19.9 19.6 .+-. 9.7 0.2 GOT (U/L) 26.5 .+-. 14.2 22.7 .+-. 4.9 0.2
CRP (mg/L) 0.9 .+-. 1.1 4.4 .+-. 9.5 0.01
[0063] As established in literature and routinely used in clinics,
there are time dependent and radiological criteria for the
classification of a non-fusion. To fulfil the definition of a
non-fusion process, the patients have to meet one or more of the
following criteria.
[0064] Time Dependent Criteria:
[0065] An incomplete vertebral body fusion after at least 24
post-operative weeks based on the presence of scar tissue formation
in the intervertebral zone. (Zenya Ito et al., SPINE Volume 35,
Number 21, pp E1101-E1105; 2010)
[0066] Radiological+Clinical Criteria:
[0067] 1) no bridging of the trabecular bone connecting the two
vertebral bodies,
[0068] 2) angular motion in excess of 5 degrees
[0069] 3) sagittal translation in excess of 3 mm
[0070] 4) radiolucencies that involve more than half of the
interfaces between the dowels and the host vertebral end plates
[0071] Based on this former classification, the non-fusion patients
showed differences in immune and inflammatory parameters in
peripheral blood. Particularly, the spinal non-fusion was strongly
associated with a significantly enhanced frequency (>2 fold) of
terminally differentiated CD8+ effector T cells expressing the
phenotype CD45+CD3+CD8+CD28- and/or CD45+CD3+CD8+CD57+(CD8+
TEMRA).
[0072] Accordingly, patients with enhanced levels of CD8+ TEMRA
cells, because of a personal history of chronic immunostimulation
(e.g. by persistent and frequently reactivating viruses like
CMV/EBV), have a worse vertebral body fusion or quality following
spinal fusion surgery as a result of an overwhelming inflammation
that inhibits normal vertebral body fusion.
[0073] The data presented herein demonstrate that the individual
immune profile (frequency of CD8+ TEMRA) is a reliable biomarker
for predicting impaired bone fusion patients allowing early
interventions with known and established methods.
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